Passaging and harvesting formulation and method for human pluripotent stem cells

ABSTRACT

Formulations and methods are disclosed for the harvesting and subsequent passaging of human pluripotent stem cells without the use of enzymes and/or scraping to dislodge cells from cell culture vessels. The formulations and methods permit the harvesting of cells as large clusters from the surface of various cell culture vessels including multilayer cell culture vessels. Further, the formulations and methods provide high yields of harvested cells for subsequent passaging and high post-harvest cell viability. Pluripotent stem cells passaged with the formulations according to the methods remain undifferentiated and express typical stem cell markers, while, at the same time, they retain the differentiation capability and are able to differentiate into the cells in all three germ layers and generate teratomas, even after numerous rounds of harvesting and passaging. These hPSCs also maintain normal karyo-type after passaged with the formulations for extended period of time.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of PCT/US2012/038321, filed on May17, 2012, which claims the benefit of priority to U.S. Application No.61/487,087, filed on May 17, 2011, the entire contents of each of whichare hereby incorporated in total by reference.

FIELD OF THE INVENTION

The present invention relates to a formulation and method forharvesting/passaging pluripotent stem cells. Specifically, the presentinvention relates to 1/ formulations including sodium citrate and amethod of use thereof; 2/ methods of identifying formulations based onthe Ca²⁺ chelator concentration and osmolarity; and 3/ use of suchformulations.

BACKGROUND OF THE INVENTION

Human pluripotent stem cells (hPSCs), including human embryonic stemcells (hESCs) and induced pluripotent stem cells (iPSCs), canproliferate indefinitely in culture while maintaining the capability todifferentiate into multiple types of somatic cells. These cells aregreatly valued as providing unlimited cell source in cell therapy andregenerative medicine. As demonstrated by recent FDA approval ofclinical trials, human embryonic stem cell (hESC)-based cell therapiesare progressing from bench to clinic. However, currently available plateand T-flask-based culture platforms severely limit the scalability ofhPSCs production to commercially relevant lot sizes. To unleash thepotential of hPSCs in cell therapy and regenerative medicine, a scalablehPSC manufacturing process must be developed. Scaling up existingflask-based processes is a critical stepping stone in translatingcurrent hPSC research into clinical application. One of the biggestchallenges is to establish a scalable passaging method for large scalemultilayer vessels that maintains high yield, pluripotent phenotype, andkaryotypic stability.

Traditionally, hPSCs are harvested and passaged as colony fragments bymechanical scraping with or without pre-treatment with enzymes (such ascollagenase or Dispase®). This process is labor intensive and cannot beapplied in culturing hPSCs in multilayer cell culture vessels, theplatform widely used in producing commercial scale adherent cells. Cellsgrowing in multilayer cell culturing vessels cannot be accessed forscraping. In addition, mechanical scraping causes damage to cells.Without scraping, cell viability can increase up to 90 percent. Knownmethods related to single-cell passaging and harvesting are notdesirable due for example, to concerns related to low post-passaging andcryopreservation recovery and abnormal karyotype, associated with lowcloning efficiency of hPSCs. Although Rho-associated kinase inhibitors(ROCK Inhibitors) have been reported to be able to improve hPSC cloningefficiency, the mechanism is not fully understood and the effect of ROCKInhibitors on hPSC culture is yet to be evaluated. Therefore, passaginghPSCs as single cells in the presence of ROCK Inhibitors is not widelyaccepted.

Recently, passaging hESCs with non-enzymatic cell detachment solutions,mainly EDTA (ethylene diamine tetraacetic acid) solutions, has beenadopted by some hESC labs and is spreading from academic labs intoindustry. One of the commercially available EDTA-containing solutionsfor cell dissociation is Versene® EDTA, which contains 0.55 mM EDTA andhas been used for harvesting and passaging hPSCs. The typical procedureof passaging hESCs with Versene® EDTA starts with washing the culturewith Ca²⁺/Mg²⁺-free buffer (for example, Dulbecco's phosphate-bufferedsaline; DPBS), followed by incubating the culture in Versene® EDTA for4-9 minutes. Versene® EDTA is then removed and cells are physicallyremoved from the surface as clusters by manual hosing of the cells withculture medium via pipetting. Compared with the conventionalenzymatic-treatment-followed-by-scraping method (see Table 1), theadvantage of this method is that (1) it uses a non-enzymaticsolution—thus, there is no need for post-detachment washing orcentrifugation to eliminate enzyme, and (2) it does not requirescraping—the cells treated with Versene® EDTA can be washed off thesurface. As described in Table 1, and illustrated in FIG. 1, comparedwith hESCs treated with enzyme and scraped off the culturing surface,the hESCs treated with Versene® EDTA and detached without scraping havehigher post-detachment viability and re-attach to the new culturingsurface much faster (minutes vs. hours) when passaged.

TABLE 1 Methods of Harvesting/Passaging hESCs Conventional Enzymatic andVersene ® EDTA Scraping Method Method 1. remove culture medium 1. removeculture medium 2. incubate in collagenase or 2. wash once withCa²⁺/Mg²⁺- Dispase ® at 37° C. for 2-5 minutes free buffer (for example,DPBS) 3. remove collagenase or Dispase ® 3. incubate in Versene ® EDTAat room temperature for 4-9 minutes 4. wash three times with culture 4.remove Versene ® EDTA medium 5. scrape hESCs off the surface in 5. hosethe cells off the surface culture medium with cell scraper or withculture medium pipette tip 6. collect the colony clumps (harvest) 6.collect the cell clusters (harvest) or transfer into a fresh culturevessel or transfer into fresh culture vessel (passage) (passage)

However, when applied into expanding hESC in multilayer vessels, theVersene® EDTA passaging/harvesting method is not ideal. Versene® EDTAseems to breaks down cell-cell association faster than it breakscell-surface bonding. If hESC culture is over-treated with Versene® EDTA(>9 minutes), a greater percentage of cells come off the surface assingle cells rather than clusters or clumps. In order to avoid gettingtoo many single cells, the treatment time normally is controlled betweenseven to nine minutes. After the removal of Versene® EDTA, in six-wellplate or T-flask culture format, fluidic shear force generated by hosingwith culture medium via manual pipetting is needed to dislodge the cellsoff the surface. However, hESC culture in multilayer vessels cannot bemanually sheared with culture medium as pipettes cannot be introducedinside the vessels. Instead, in this culture format, after Versene® EDTAis replaced with culture medium, vigorous tapping is applied to dislodgethe cells. The mechanical force (tapping) has to follow the replacementof Versene® EDTA with culture medium immediately because Versene® EDTAtreated hESCs quickly re-attach to the surface once they come in contactwith culture medium. In fact, with the current state-of-art, it is onlypossible to harvest 40-70% of the entire culture in multilayer cellfactories—dramatically impacting the yield of these very expensivecells. One possible solution to increase the yield is not to replaceVersene® EDTA with culture medium and to dislodge the cells in Versene®EDTA instead. However, in this case, the exposure time of cells toVersene® EDTA is increased, which increases the risk of getting too manysingle cells and obtaining karyotypic unstable colonies. In addition,extra steps of post-detachment processing have to follow to remove orneutralize Versene® EDTA from the final harvest, which will not only addto the labor intensity but also further the breakup of the small hESCclusters.

There is therefore a need for a scalable and high-yielding passaging andharvesting formulation and method for hPSCs that eliminates or reducesthe drawbacks of methods known in the art.

SUMMARY OF THE INVENTION

The present invention provides a non-enzymatic reagent formulation and amethod of harvesting and subsequently passaging pluripotent stem cellsas clusters with high yield and high post-detachment cell viability.

It is an object of the invention to provide a scalable and high-yieldingpassaging and harvesting formulation and method for hPSCs thateliminates or reduces the drawbacks of methods known in the art.

It is a further object of the invention to provide a formulationcomprising sodium citrate.

It is a further object of the invention to provide a formulation andmethod optimized for harvesting and passaging hPSCs in reference to hPSCparameters such as high viability, high yield, large post-detachmentcluster size, serial passageability, and maintenance of the pluripotentphenotype (for example, expression of markers typically associated withstem cells such as OCT4, Sox2, Nanog, SSEA4, TRA-1-60 and TRA-1-81) andkaryotypic stability.

It is a further object of the invention to provide a formulation andmethod that can be used in any hPSC lab as routine lab practice toexpand hPSC cultures with reduced labor intensity and process time.

It is a further object of the invention to provide a formulation andrelated method that does not require mechanical scraping to remove cellsfrom the surface of the culture vessel.

It is a further object of the invention to provide a formulation andrelated method wherein the harvested cells do not need to be washed andcentrifuged to remove the agents used to detach the cells from thesurface of the culture vessel.

It is a further object of the invention to provide a formulation andmethod, whereby over 90% of hPSCs grown in multilayer cell culturevessels can be harvested with over 90% viability.

It is a further object of the invention to provide a closed and scalablehPSC manufacturing process through culturing hPSCs in multilayer cellculture vessels and by washing, concentrating, vialing andcryopreserving cell harvest with automatic downstream processingtechnology.

It is a further object of the invention to provide a process ofexpanding and passaging hPSCs from T-flasks into multilayer cellfactories with a novel non-enzymatic harvesting and passaging method,followed by downstream processing with continuous counter-flowcentrifugation technology (for example, kSep® technology).

It is a further object of the invention to provide a method ofdeveloping a cell-detaching solution for hPSCs wherein the sizedistribution of the detached clusters and the percentage of the culturedetached at given treatment time can be controlled with the osmolarityand Ca²⁺ chelator concentration.

It is a further object of the invention to provide a method forharvesting and subsequent passaging of hPSCs grown in suspension asaggregates or on microcarriers that includes incubating the hPSCaggregates or hPSCs on microcarriers, in either a formulation disclosedherein or a formulation identified by a method disclosed herein, in cellculture vessels for two to twenty minutes allowing the hPSC aggregatesto disintegrate or to allow the hPSCs to detach from the microcarriers,with cell viability between about 85-100 percent.

It is a further object of the invention to provide a method forharvesting and subsequent passaging of hPSCs, where the hPSCs arepassaged with a high split ratio (1:10 to up to 1:60; or density ofcells at seeding of about 30E3/cm² to as low as 5E3/cm²) and the culturereaches confluence within seven days after split.

It is a further object of the invention to provide a method forharvesting and subsequent passaging of hPSCs where the hPSCs maintainpluripotency and normal G-banding karyotype at over 50 passages.

It is a further object of the invention to provide formulations, andmethod of use thereof, providing for selectively detaching and passagingundifferentiated hPSCs.

It is a further object of the invention to provide a means forharvesting and subsequent cryopreserving hPSCs with high post thawrecovery and re-plating efficiency.

It is a further object of the invention to provide a method ofdownstream processing of harvested hPSC clusters in a closed systemincluding continuous counter flow, centrifugation, formulation,automated vialing and cryopreservation with controlled rate freezer.

Accordingly, in one embodiment, a formulation is provided for harvestingand subsequent passaging of human pluripotent stem cells withoutscraping and without substantial loss of viability. In one aspect of theembodiment, the formulation includes, for example, sodium citrate, asalt, and a phosphate-buffered saline solution, at an osmolarity ofabout 250-1050 mOsmol/Liter. In an alternative aspect the osmolarity is311-1014 mOsmol/Liter. In another aspect of the embodiment, theformulation is used to harvest and passage embryonic stem cells andinduced pluripotent stem cells. The concentration of citrate is, forexample, about 0.15 to 150 mMol/Liter. The salt is, for example, NaCl,KCl, Na₂HPO₃, NaH₂PO₃, K₂HPO₃, or KH₂PO₃. When the salt is KCl, theconcentration is, for example, about 1.00-1400 mMol/Liter Alternatively,when the salt is KCl, the concentration is about 1.35-1350 mMol/Liter.In an aspect of the embodiment, the osmolarity of the formulation is,for example, about 400-700 mOsmol/Liter. In another aspect of theembodiment, the osmolarity of the formulation is, for example, about418-570 mOsmol/Liter. In another aspect of the embodiment, theformulation is pH buffered with, for example, bicarbonate, phosphate,ethanolamine, triethanolamine, or trometamol. The pH of the formulationis, for example, about 7-8, In an alternative embodiment the pH is about7.2-7.8. In one aspect the phosphate-buffered saline solution is, forexample, Ca²⁺/Mg²⁺-free Dulbecco's phosphate buffered saline (DPBS). Insome aspects of the embodiment, treatment of the cells with theformulation results in the harvest of, for example, at least 90% of thecells from the surface of the culture vessel and cell viability of, forexample, at least 90%.

In another embodiment, a method is provided for producing a formulationfor harvesting and subsequent passaging of human pluripotent stem cellswithout scraping and substantial loss of viability.

In another embodiment, a method is provided for harvesting andsubsequent passaging of hPSCs comprising incubating the hPSCs in one ofthe formulations described in the preceding paragraphs and in cellculture vessels for a period of time to allow the hPSCs to detach fromthe cell culture vessels in clusters with high yield and highpost-detachment cell viability of, for example, about 85-100%. In oneaspect of the embodiment, the cell culture plates or vessels are, forexample, petri dishes, multi-well cell culture plates, stacked cellculture apparatus, multilayer cell culture factories, and similarvessels known in the art to be capable of supporting the culture ofhPSCs. In some aspects of the embodiment, the cells are treated with theformulation for about 2-20 minutes. In one embodiment, the treatmenttime is 5-15 min for hPSCs cultured in mTeSR1®. In another embodiment,the treatment time is 8-20 min for hPSCs cultured in StemPro®. (StemPro®and mTeSR1® are examples of commercially available defined media forhPSC culture.) In aspects of the embodiment, the cells are harvested inclusters with sizes ranging, for example, from about 10-1000 μm. Moreparticularly, the cluster size is, for example, about 40-500 μm. In someaspects of the embodiment, the method results in the harvest of, forexample, at least 90% of the cells from the surface of the culturevessel and cell viability of at least 90%.

In another embodiment additional passaging and harvesting formulationsare provided including formulations containing EDTA and EGTA, other Ca²⁺chelators besides sodium citrate, or combinations of various Ca²⁺chelators. Two factors are identified related to cell detachment, theCa²⁺ chelator concentration and osmolarity.

In another embodiment, a method of developing a cell-detaching solutionfor hPSCs is provided wherein the size distribution of the detachedclusters and the percentage of the culture detached at given treatmenttime can be controlled with the osmolarity and Ca²⁺ chelatorconcentration.

These and other objects are achieved in the present invention.

There has thus been outlined, rather broadly, the more importantfeatures of the invention in order that the detailed description thereofthat follows may be better understood, and in order that the presentcontribution to the art may be better appreciated. There are, of course,additional features of the invention that will be described furtherhereinafter.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that equivalent constructions insofar as they do not departfrom the spirit and scope of the present invention, are included in thepresent invention.

For a better understanding of the invention, its operating advantagesand the specific objects attained by its uses, reference should be hadto the accompanying drawings and descriptive matter which illustratepreferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS AND THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates the percent viability of hESCs detached from aculturing surface following enzymatic treatment and scraping vs.non-enzymatic treatment and non-scraping.

FIG. 2 illustrates EDTA/EGTA detachment vs. sodium citrate detachment.

FIG. 3 illustrates hESCs continuously passaged with sodium citrateshowing typical hESC colony morphology and growth.

FIG. 4 illustrates the normal karyotype of hESCs passaged five timesfollowing harvesting with sodium citrate.

FIGS. 5a-e illustrate harvest of hESC culture in two-layer multilayercell stacks (sodium citrate vs. EDTA).

FIG. 6 illustrates optimization of sodium citrate formulation for hESCdetachment.

FIG. 7 illustrates the density increase of viable hESCs post-KSepprocessing (volume reduction) and the viability change before and afterapplication of continuous counter-flow centrifugationtechnology)(kSep®).

FIG. 8 illustrates the recovery of cells after cryopreservation.

FIG. 9 illustrates re-plating of hESCs post-continuous counter-flowcentrifugation technology (kSep®) and post cryopreservation.

FIG. 10 illustrates the trend of increasing detachment with the increaseof treatment time with various dilutions of sodium citrate formulationin MEF-CM.

FIG. 11 illustrates the trend of increasing detachment with the increaseof treatment time with various dilutions of sodium citrate formulationin mTeSR1®.

FIG. 12 illustrates the effects of osmolarity and potassiumconcentration on the ability of sodium citrate formulations to promotehESC detachment.

FIG. 13 illustrates the osmolarity and final concentrations of sodiumcitrate and KCl of various formulation solutions used in the experimentsdescribed in FIG. 14A-C.

FIGS. 14A-C illustrate the effect of osmolarity on the ability of sodiumcitrate formulations to promote hESC detachment and maintain largecluster size.

FIGS. 15A-C illustrate the effects of sodium citrate concentration onhESC detachment and on size of detached clusters.

FIGS. 16A-B illustrate the effects of EDTA, EGTA, and sodium citrate onhESC detachment.

FIG. 17 illustrates formulations used in time course studies.

FIG. 18A illustrates the effect of treatment time with variousformulation solutions on hESC detachment;

FIG. 18B illustrates the size distribution of the hESC clusters detachedby treatment of various sodium citrate formulations, and the effect ofthe treatment time on the cluster size distribution.

FIG. 19 illustrates the plating efficiency of hESC clusters generatedusing Versene® EDTA and various sodium citrate formulations, and theeffect of treatment time on the plating efficiency.

FIGS. 20A-B illustrate determination of the operational window (that is,time frame of treatment) to achieve optimal harvest and cluster sizeusing cell detachment solutions.

FIGS. 21A-B illustrate the comparison of the operational windows and theplating efficiency of hESC clusters detached using sodium citrateformulation solution with that of the clusters detached using EDTA andEGTA at high concentrations

FIG. 22 illustrates the experimental procedure for harvesting hESCs from6-well-plates using either Versene® EDTA or sodium citrate formulationsolution when EDTA or sodium citrate is removed during incubation (“DryPassaging” methodology).

FIG. 23 illustrates the chemical compositions, performancecharacteristics, and potential applications of three sodium citrateformulation solutions.

FIG. 24 illustrates the leave-in ‘passage” methodology.

FIG. 25 illustrates the number of viable cells harvested andpost-detachment viability of hESCs following “leave-in passage” withsodium citrate formulation solutions.

FIG. 26 illustrates the cluster size distribution, plating efficiencyand the morphology of re-plated hESC colonies following “leave-inpassage” with sodium citrate formulation solutions.

FIG. 27 illustrates the effects of Versene® EDTA and various sodiumcitrate formulation solutions on passaging of mesenchymal stem cells(MSC).

FIGS. 28A-C illustrate optimization of sodium citrate formulationstowards high plating efficiency after extended treatment.

FIG. 29 illustrates the immunocytochemistry of hESC markers, asevaluation of the self-renewal of the hESCs after long-term passagingwith sodium citrate formulation.

FIG. 30 illustrates the immunocytochemistry of embryoid bodydifferentiation of hESCs, as evaluation of the differentiationcapability of hESCs passaged for a long term with sodium citrateformulation.

FIG. 31 illustrates the histology of teratomas generated inimmune-deficient mice injected with hESCs passaged for a long term withsodium citrate formulation, as further confirmation of differentiationcapability of long-term hESC culture.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Preliminary Screening and Characterization of Various Non-Enzymatic CellDetachment Formulation Solutions and Methods

Disclosed herein is a non-enzymatic reagent formulation and a method ofharvesting/passaging pluripotent stem cells as clusters with high yieldand high post-detachment cell viability (>90%). These cells can then beprocessed as final harvest or seeded into new culture vessels to befurther expanded. In one embodiment, the disclosed formulation includessodium citrate, which disrupts the cell-surface bond and cell-cellassociation by chelating/sequestering Ca²⁺, the divalent cation requiredfor cell-surface and cell-cell binding. This sodium citrate-basedformulation and process is specially designed and developed to addressthe unique challenges in routine or scale up hPSC culture andmanufacturing processes. hPSCs are normally passaged as multi-cellularclusters/aggregates, and passaging hPSCs as single-cells is to beavoided due to low cloning efficiency of hPSCs and the high risk ofkaryotypic abnormality. This formulation and method is additionallyoptimized for harvesting and passaging hPSCs in reference to the keyquality parameters of hPSCs, for example, viability, yield,post-detachment cluster size, passageability, and maintaining apluripotent phenotype. This formulation and process can be used in anyhPSC lab as routine lab practice to expand hPSC culture with reducedlabor intensity and process time. For example, this formulation andprocess does not require mechanical scraping to get the cells off thesurface and the cell harvest does not need to be washed and centrifugedto remove the agents used to detach the culture. This formulation andprocess especially benefit large-scale hPSC production when the cellsare growing in multilayer cell culture vessels where scraping cannot beapplied. Using this formulation and method, more than 90% of hPSCs grownin multilayer cell culture vessels can be harvested with more than 90%viability.

A variety of non-enzymatic cell detachment solutions at variousconcentrations were screened with one objective being to improve theyield of hESCs harvested from multilayer culture vessels while retainingthe simplicity of the Versene® EDTA harvesting/passaging method. Thisscreening included, for example, Versene° EDTA solutions at 0.1, 0.55,1, 3, and 10 mM, Versene® based ethylene glycol tetraacetic acid (EGTA)solutions at 0.1, 0.55, 1, 3, and 10 mM, and 1× sodium citrate solution(10× solution: 0.15 M sodium citrate, 1.35 M potassium chloride (KCl),diluted to 1× in Ca²⁺/Mg²⁺-free DPBS).

All of these reagents (EDTA, EGTA and sodium citrate) are Ca²⁺ chelatorsand have been used historically for detaching adherent cells in culture.As mentioned previously, Versene® EDTA has been used routinely forharvesting/passaging hESCs in some labs; both EDTA and EGTA (incombination with trypsin) were used to passage hESCs in a studypublished by Thomson et al. at Roslin Institute in Scotland in 2008(Thomson et al. (2008), “Human Embryonic Stem Cells Passaged UsingEnzymatic Methods Retain a Normal Karyotype and Express CD30”, Cloningand Stem Cells, 10 (1), 1-17.).

hESCs cultured in Murine Embryonic Fibroblast-conditioned medium(MEF-CM) were treated with solutions including those described above asfollows: (1) remove culture medium; (2) wash once with Ca²⁺/Mg²⁺ freebuffer (for example, DPBS); (3) incubate in solution at roomtemperature; (4) remove solution; and (5) soak the colonies in MEF-CMfor 0.5-1 minute to let them have a chance to stick back onto thesurface. The colonies were then hosed with culture medium to see if theycan be detached from the surface. Related to incubation step #3 above,it is noted that 4-9 minutes is the norm for Versene® EDTA. It isgenerally longer with culture grown in undefined serum or serumreplacement-containing medium (for example, MEF-CM), and shorter indefined serum-free medium such as mTeSR1®. Thus, an incubation window of2-12 minutes is contemplated. Treatment for more than 9 minutes canresult in the generation of too many single cells. Treatment for lessthan 2 minutes can be too short a time frame resulting in partialdetachment. Sodium citrate solution (1×) can detach the cells grown inmTeSR1® in 2-3 minutes, and detach the cells grown in MEF-CM in 3-4minutes.

Related to step #5, this step was executed in the initialdetachment-solution screening study to see whether the cells re-stickonto the surface in culture medium. For harvesting cells grown in anopen platform such as 6-well plates, the cells are hosed off the surfaceright after removing the cell-detachment solution. However, formultilayer cell culture vessels, after draining out the detachmentsolution, the culture medium is poured into the vessel first, the mediumis leveled in all layers, and then the vessel is tapped to dislodge thecells in the culture medium. In one embodiment, the pouring and levelingtake 0.5-1 minute in a ten-layer vessel. In the cGMP setting, additionaltime may be required. A portion of the culture is inevitably soaked inthe culture medium during these steps and may re-stick onto the surface.As mentioned previously, this is a difficulty with the use of Versene®EDTA, which results in the incomplete detachment/harvest of the culturein multi-layer cell culture vessels. Therefore, soaking the colonies inculture medium for 0.5-1 minute was done to evaluate the re-sticking ofthe cells onto the surface.

As illustrated by FIG. 2, after this procedure, 85% of the Versene®(0.55 mM EDTA)-treated culture remained on the surface (FIG. 2a ), whilenearly all the cells came off the surface in 1× sodium citrate (sodiumcitrate formulation) treated culture (FIG. 2b ). The visual assessmentof the percentage of the culture remaining on the surface aftertreatment is plotted in FIG. 2c . Through this experiment, sodiumcitrate solution is surprisingly identified as a superior reagentcompared to Versene® EDTA for harvesting hESC culture. Sodium citrateformulation disrupts the cell-surface bond to a greater extent thanVersene® EDTA and sodium citrate formulation treated hESCs do notre-stick to the surface in culture medium as fast and easily as Versene®EDTA or EGTA formulation-treated hESCs.

Two commercially available solutions were tested as potential solutionto harvest hPSCs out of multilayer cell culture vessels. One was fromMillipore, the “PBS Based Enzyme-Free Cell Dissociation Solution”, theother was “Hank's Based Enzyme-Free Cell Dissociation Solution”. Both ofthe solutions contain EDTA, Glycerol and Sodium Citrate. When used on H9hESCs cultured in MEF-CM, neither one of the solutions was able todetach the cells effectively.

Further experimentation was performed to determine whether sodiumcitrate formulation can be used to passage hESC cultures; that is,whether hESCs detached by sodium citrate formulation can be plated ontoa fresh surface to start a new culture. In one exemplary experiment,hESCs treated with sodium citrate formulation were continuously passagedsix times in MEF-CM and three times in mTeSR1®. FIG. 3 shows that hESCspassaged with sodium citrate formulation for six times in MEF-CM retaintypical hESC morphology. As illustrated by FIG. 4, the karyotype of thisculture was evaluated to be normal after five passages with sodiumcitrate formulation. Further testing has confirmed normal karyotype at10 passages in MEF-CM, 29 passages in mTeSR1®, and 25 passages inStemPro®. It is contemplated that a normal karyotype will be present atgreater than 50 passages.

Sodium citrate formulation was tested for harvesting hESCs in two-layercell stacks. This sodium citrate harvest was compared with the harvestusing Versene® EDTA. FIG. 5 provides images of the cells remaining onthe surface after harvest. The cultures in FIGS. 5a and 5b were bothharvested using Versene® EDTA, but were handled by different operators(with the strength applied to tapping of stacks to dislodge cellsvarying between the operators). The operator with stronger tappingharvested ˜70% of the entire culture, while the operator with weakertapping only harvested ˜45% of the culture. However, both the operatorsharvested more than 90% of the entire culture from the surface whensodium citrate formulation was used for the harvest (FIG. 5c ). Thepercentage of the culture detached from each two-layer stack is plottedin FIG. 5d . The number of cells harvested from each vessel was countedand the viability of the harvest was calculated (FIG. 5e ). Comparedwith Versene® EDTA, the counts of the cells harvested with 1× sodiumcitrate solution have much tighter distribution (2.64E8±5.1E7 withVersene® EDTA vs. 3.46E8±1.81E7 with sodium citrate, (see FIG. 5e ),which contributes to a more robust and consistent manufacturing process.The yield of sodium citrate formulation-harvested hESC culture was 31%higher than that of Versene® EDTA harvested culture. There was lessoperator-to-operator variance when sodium citrate formulation was used.This may be because sodium citrate formulation disrupts the cell-surfacebond more than Versene® EDTA and much less tapping effort was needed todislodge the cells.

Additional studies to further optimize the formulation and method wereconducted. In one embodiment, the optimal range for the concentration ofsodium citrate for hESC detachment was 0.1×-3× (see Table 2 for theconcentrations of sodium citrate and KCl), or higher. Surprisingly,sodium citrate formulations at higher sodium citrate concentrations didnot necessarily detach the cells faster or break the colonies up intosingle cells more than lower concentrations (FIG. 6b ). In fact, whenworking on hESCs grown in MEF-CM, sodium citrate at higher concentration(1×-3× and higher) tended to lift the hESC colonies up as whole sheets.The size of the detached hESC clump was dependent on the dilution ofsodium citrate formulation more than treatment time (1-11 minutestested). This adds to the advantage of sodium citrate formulation overVersene® EDTA, which breaks the hESC colonies into undesirable singlecells at extended treatment (>9 minutes). The clump size of the detachedhESC grown in mTeSR1® was overall smaller than that grown in MEF-CM. Theeffect of cell-detachment formulation on the size of detached clusters(clumps) of hESCs was further investigated. As described below, theosmolarity of the formulation affects the size of the clusters. Incertain embodiments, a clump size of 10-1000 μm is preferred. Inalternative embodiments, a clump size of 40-500 μm is desired. Theoperational time window for sodium citrate formulation treatment onhESCs was wider than Versene® EDTA treatment on hESCs, which isparticularly beneficial in cGMP-compliant production when productquality consistency is regulated and deviation is to be minimized Theoperational time window is further described below.

In exemplary experimentation presented herein, the solution included:sodium citrate and KCl diluted in Ca²⁺/Mg²⁺-free DPBS. It is, however,reasonably contemplated that alternative calcium chelators at optimizedconcentrations in formulations with optimal osmolarity will provideattractive results. Similarly, citrate formulations containing a saltother than KCl are contemplated. Such salts include, for example, NaCl,Na₂HPO₃, NaH₂PO₃, K₂HPO₃, KH₂PO₃, and the like.

In one embodiment, sodium citrate concentration has a preferred workingrange of 0.15-150 mM. In one embodiment for harvesting cells grown inculture medium such as MEF-CM (Murine Embryonic Fibroblast-ConditionedMedium), sodium citrate concentration has a working range of 1.0-50 mM.In an alternative embodiment, as illustrated in FIG. 6, sodium citrateconcentration has a working range of 1.5-45 mM. In one embodiment,sodium citrate concentration for passaging cells grown in MEF-CM is aworking range of 1.5-15 mM. In one embodiment, sodium citrateconcentration for harvesting cells grown in culture medium such asmTeSR1® is a working range of 1.5-30 mM. Excipients in the formulationinclude, but are not limited to, Ca²⁺/Mg²⁺-free DPBS and KCl (1.35-1350mM). The working osmolarity of this formulation is from 31.1-2050mOsmol/L. In one embodiment, the working range is 290-1015 mOsmol/L. Inanother embodiment, the working range is 299-781 mOsmol/L. In yetanother embodiment, the range is 548-781 mOsmol/L. In Table 2, sodiumcitrate solution of 10× contains 1350 mM KCl and 150 mM sodium citratein water. A series of dilutions was made by diluting the 10× solution inDPBS (without Ca²⁺ or Mg²⁺).

TABLE 2 Formulations of Sodium Citrate Solution Dilution Factor 0.1x0.3x 0.7x 1.0x 1.3x 2.0x 3.0x 10x KCl 13.5 mM 40.5 mM 94.5 mM 135 mM175.5 mM 270 mM 405 mM 1350 mM Sodium  1.5 mM  4.5 mM 10.5 mM  15 mM 19.5 mM  30 mM  45 mM  150 mM Citrate

Osmolarity of the screened cell detachment solutions was measured (Table3). Sodium citrate solution in general has a higher osmolarity than allthe Versene® -based EDTA and EGTA solutions (solutions screened in FIG.2). This may be due to the high concentration of KCl and otherexcipients in the solutions. High osmolarity of the sodium citrateformulation is identified herein as one attribute that contributes tothe unique cell detachment behavior of the solution, which is furtherdiscussed hereinafter. Table 3 illustrates osmolarity of varioussolutions.

TABLE 3 Osmolarity of Non-Enzymatic Detachment Solutions 0.1 mM 0.55 mM10 mM EDTA EDTA 1 mM EDTA 3 mM EDTA EDTA Versene ®-Based EDTA solutionsmOsmo 273 272 281 287 305 Versene ®-Based EGTA solutions mOsmo 282 282283 290 311 Sodium Citrate (Diluted in DPBS) 0.1x 0.3x 0^(.7)x 1.0x ¹.3x2.0x 3.0x 10x mOsmo 311 352 432 499 587 741 1014 >2050

Continuous counter-flow centrifugation technology can be used toconcentrate and wash hESC harvest. As illustrated in FIG. 7, hESC (grownin MEF-CM) harvest is five times concentrated with 10% drop in viabilityafter processing with kSep®. hESCs processed with kSep® remain asclusters and plate well with 10% drop in plating efficiency, comparedwith pre-kSep® fresh harvest (FIG. 9). hESCs formulated and vialed withM-1 filling machine cryopreserve efficiently in CryoStor10 with morethan 90% viable cell recovery and 50% post-thaw plating efficiency(FIGS. 8-9).

As further discussed in EXAMPLE 11, in one embodiment, the process ofexpanding and passaging hESCs grown in mTeSR1® from T-flasks intomultilayer cell factories is optimized with a novel non-enzymaticpassaging method, followed by downstream processing with continuouscounter-flow centrifugation technology.

In another embodiment, hESC culture in MEF-CM is expanded, with Versene®EDTA passaging, from six-well plate into T-flasks, and further intomultilayer cell culture vessels. The final harvest from multilayer cellfactories was characterized with flow cytometry and more than 90% ofthese cells expressed pluripotency markers including OCT4, SSEA4,Tra-1-60, and Tra-1-81. The final cell harvest was concentrated morethan 14 times after automated downstream processing, with only 2% dropin cell viability.

Further studies related to treatment time and its correlation with thedilutions of the citrate formulation were conducted and are furtherdefined below in Table 4. FIGS. 10 and 11 demonstrate the effect of thedilution of sodium citrate formulation on the trend of culture harvestincrease with the increase of treatment time. The study illustrated inFIG. 10 was conducted on hESC culture grown in MEF-CM, and the studyillustrated in FIG. 11 was conducted on hESC culture grown in mTeSR1®.

TABLE 4 in MEF-CM in mTeSR1 ® treatment time window Sodium 0.1X 8 min-10min Citrate 0.3X 4 min-11 min 6 min-10 min dosage 0.7X 3 min-11 min 4min-10 min  1X 3 min-11 min 3 min-10 min 1.3X 3 min-11 min 2 min-10 min 2X 3 min-11 min 3 min-10 min  3X 5 min-11 min minimal treatment time (min) to achieve 85% detachment Sodium 0.1X 8 Citrate 0.3X 4 6 dosage0.7X 3 4  1X 3 3 1.3X 3 2  2X 3 3  3X 5

It is contemplated that with the sodium citrate formulations the cellscan be harvested from a variety of surfaces including but not limited toMatrigel-coated surface, gelatin-coated and MEF-plated surface, and/orchemically defined surfaces. Examples include, for example, syntheticplastic or peptide-bound surfaces (Corning Synthemax and Nunclon (Nunc)Vita surfaces), surfaces coated with defined recombinant or nativeproteins (e.g. vitronectin, fibronectin, laminin and collagen). Thereare two widely-used surfaces for hPSCs: the MEF-seeded surface (MurineEmbryonic Fibroblasts are seeded onto surface pre-coated with gelatin),and Matrigel-coated surface (Matrigel is an extracellular matrix derivedfrom mouse tumor). Both Synthemax and Nunc Vita are defined surfacesavailable on the market and marketed as a surface that can be used forhPSC culture. Sodium citrate formulations can be used to harvest hPSCsgrown in various types of culture medium, including, but not limited to,regular culture medium used for culturing hPSCs on MEF-seeded surfaces,MEF-CM, mTeSR1® and StemPro®.

EXAMPLE 2 All the Studies in Example 2 were Conducted on hESC Culturesin mTeSR1®

Determination of Optimum Sodium Citrate Formulation

Comparative Effects of Osmolarity, Potassium, and Sodium on the Abilityof Sodium Citrate to Promote Cell Detachment

The 1× sodium citrate formulation described above is 15 mM sodiumcitrate, 135 mM KCl, pH 7.31, and 499 mOsmol/L in DPBS. Further studieswere performed to determine the effects of osmolarity, KCl on theability of the sodium citrate formulation to promote cell detachment.FIG. 12 shows the percentage of detachment of hESC in cultures treatedwith various solutions of sodium citrate. The results indicate that theosmolarity of the solution and the concentration of sodium citrate areimportant factors for achieving optimal cell detachment. In addition, atnearly normal osmolarity, the addition of potassium (KCl) to the sodiumcitrate formulation caused greater percentage detachment than theaddition of sodium (NaCl) (compare solution #4 to solution #5)suggesting that KCl may be a better choice of salt in this formulationthan NaCl. Not to be limited by theory, one possible explanation forthis finding is that the additional sodium (Na⁺) inhibits theelectrolysis of sodium citrate and consequently lowered the availabilityof the Ca²⁺ chelating citrate ions.

Effect of Osmolarity on the Ability of Sodium Citrate to Promote CellDetachment and Maintain Viability

Studies were performed to determine the effect of osmolarity on theability of sodium citrate to promote hESC detachment. Sodium citratesolutions/formulations of various osmolarity were prepared by adjustingthe concentration of KCl and maintaining a constant concentration ofsodium citrate (see FIG. 13). As shown in FIG. 14A, high (andapproximately the same) viability was observed with all solutionswhereas maximum harvesting of cells was achieved after treatment ofcells for 5 minutes with sodium citrate solutions (15 mM citrate) havingan osmolarity of between 299-781 mOsmol/L. Substantially reducedharvesting was observed using a sodium citrate solution with anosmolarity of 169 mOsmol/L and an osmolarity of 1016 mOsmol/L suggestingthat there is a threshold between about 299-781 mOsmol/L at which highpercentage of culture could be harvested. In fact, microscopic images(FIG. 14B) revealed substantially larger clusters of harvested cellsfrom cultures treated with 781-1016 versus 169-548 mOsmol/L solutions ofsodium citrate. The images of the detached clusters were analyzed andthe sizes of the clusters were quantified. The distribution of thecluster sizes is shown in FIG. 14C. As demonstrated in images and thesize distribution plot. The percentage of the culture harvested as largecell clusters was increasing with the increase of the osmolarity. Therewas a substantial increase in the measured sizes of the detachedclusters obtained from cultures treated with 781-1016 mOsmol/L solutionsversus 169-548 mOsmol/L solutions (mostly 250-900 equidiameter sizeclusters versus mostly 40-150 equidiameter size clusters, respectively).

Effect of Sodium Citrate Concentration on Cell Detachment and Viability

Studies were performed to determine the effect of citrate concentrationon hESC detachment and viability. Sodium citrate solutions of variousconcentrations were prepared at about 265 mOsmol/L and a pH of about7.2. As shown in FIG. 15A, high (and approximately the same) viabilitywas observed with all solutions whereas the percentage of harvestedcells increased with increasing concentrations of up to about 30-45 mMsodium citrate. Only slightly improved harvesting was achieved using 75mM sodium citrate. However, there appeared to be a threshold betweenabout 30-75 mM sodium citrate at which “sheeting harvest” (hESC coloniesdetached as big clusters or sheets of cells) occurred. In fact,microscopic images revealed substantially larger clusters of harvestedcells from cultures treated with 30-75 mM sodium citrate solutions ofsodium citrate (FIG. 15B). There was a corresponding increase in themeasured sizes of clusters obtained from cultures treated with 30-75 mMsodium citrate solutions versus 1.5-15 mM sodium citrate solutions(mostly 250-900 equidiameter size clusters versus mostly 40-150equidiameter size clusters, respectively) (FIG. 15C).

Comparative Effects of EDTA, EGTA, and Sodium Citrate on Cell Detachment

Studies were performed to determine the comparative effects of chelatorson hESC detachment. The Ca²⁺ chelation capacity of citrate, EDTA, andEGTA are 1:1.5, 1:1, and 1:1, respectively, based on stoichiometry. EDTAalso binds Mg²⁺ and other metals whereas EGTA is more specific forbinding of Ca²⁺. Cell detachment achieved by a solution of sodiumcitrate (15 mM sodium citrate at 400 mOsmol/L (achieved by addition ofKCl) and a pH of 7.8) was compared to that achieved by 22.5 mM and 45 mMsolutions of EDTA and EGTA at 400 mOsmol/L and a pH of 7.8. As shown inFIG. 16A, EDTA and EGTA at concentrations higher than 22.5 mM detachedhESC colonies as effectively as sodium citrate. The measured sizes ofredistributed clusters obtained from cultures treated with the varioussolutions were similar (mostly 40-200 equidiameter size clusters) (FIG.16B).

EXAMPLE 3

Time Course Studies

Versene® EDTA Versus Sodium Citrate

Studies were performed to determine the operational time windows thatprovide good percentage detachment and cluster size distribution. Thedetachments in the studies described above were evaluated after fiveminute treatment with cell detachment formulations. Here, treatment timeis extended up to 20 minutes, and detachment and cluster size areexamined under various time points (2, 5, 8, 10, 15 and 20 minutes).Formulations of moderate osmolarity and pH are selected from priorstudies and the time span (operational window) of treating hESC culturewith these formulations that would yield a desirable percentagedetachment and post-detachment cluster size distribution is determinedCell cultures were treated with the solutions of FIG. 17 for 2, 5, 8,10, 15 or 20 minutes and then tapped twice to dislodge the cells. Foreach treatment time point, cultures were evaluated for percentagedetachment of cells and cluster size in order to determine the optimaloperational window. Cell viability and plating efficiency of detachedcells were evaluated at the 5, 10, and 20 minute time points. FIG. 18Ashows the effects of Versene EDTA and various sodium citrate solutionson hESC percent detachment at each treatment time point. FIG. 18B showsthe effects of Versene® EDTA and various sodium citrate solutions oncluster size distribution at each treatment time point. FIG. 19 showsthe effects of Versene® EDTA and various sodium citrate solutions on theplating efficiency of detached cells at each treatment time points of 5,10 and 20 minutes.

Based on the results obtained from this study, and not to be limited bytheory, an operational window similar to that depicted in FIG. 20A canbe established and appropriate parameters can be selected to achieve adesired percentage of cell detachment and cluster size (equi-diameter).For example, as shown in FIG. 20B the criteria used to determine theoperational window is (1) the percentage of culture detached is over 70%and (2) the percentage of culture detached as 40-500 um clusters is over90%. As indicated by the experiment data, in general, the percentdetachment increase with the increase of the treatment time and thecluster size decrease with the increase of the treatment time. Based onthese plots, treating hESC culture with citrate formulation Dis2#3 for3.5 to 7 min (operational window) would result in detaching >70% of theculture as mostly (>90%) 40-500 um clusters. The operational windowsdetermined under the same criteria for other citrate formulations andVersene® EDTA are narrower. In fact, based on the plot, to meet the samecriteria with Versene® EDTA, the culture needs to be treated for exactly10 min (0 duration). Wider operational window, which gives moreoperational flexibility without affecting the consistence of productcharacteristics, is more desirable, especially in cGMP production. Basedon FIG. 19, plating efficiency drops with the increase of the treatmenttime and longer than 10 min treatment time is not desirable because ofthis. Accordingly, best practice (based on high percent detachment,desirable cluster size range and high plating efficiency) for detachinghESCs with citrate formulation Dis2#3 would be 5-7 min treatment, and 10min treatment with Versene® EDTA. At their respective optimal treatmenttimes, Versene® EDTA harvested over 20% fewer cells than sodium citrate.

High Concentration of EDTA and EGTA

Following the same basic approach as used above for determining theoperational windows for various sodium citrate formulations and Versene®EDTA, experiments were performed to determine the operational windowsand plating efficiency for 22.5 mM Versene® EDTA and 22.5 mM EGTAformulations described in EXAMPLE 2, as comparison to Versene® EDTA andsodium citrate (formulation Dis2#3). The operational windows weredetermined based on the criteria of “>70% of the culture is harvestedand >90% of the cells harvested are in the format of 40-500 μm cluster”.As shown in FIG. 21A, the durations of the operational windows of 22.5mM EGTA and Versene® EDTA formulations are similar to that of citrateformulation Dis2#3, and are much wider than that of Versene® EDTA. Thestarting time points of the operational windows of 22.5 mM EGTA and EDTAformulations are the same, and are earlier than that of citrateformulation Dis2#3. However, as shown in FIG. 21B, hESCs detached with22.5 mM EGTA and EDTA formulations did not re-plate well (5, 15 and20min treatment time points examined), and sodium citrate (solutionDis2#3) substantially outperformed high-concentration (22.5 mM) EDTA andEGTA formulations in terms of cell plating efficiency post-harvest(plotted are plating efficiencies at 5 minute treatment time pointnormalized with that of citrate formulation Dis2#3).

It is demonstrated herein that sodium citrate formulations outperformEDTA-containing and EGTA-containing formulations in passaging hPSCs.Furthermore, another advantage of sodium citrate over EDTA and EGTA isthat leftover sodium citrate should have less deleterious effects oncell cultures than EDTA or EGTA. (Sodium citrate is sometimes used as anormal component in various culture media of various cell types.) Forexample, as seen in EXAMPLE 6, sodium citrate formulation solutions donot necessarily need to be removed after treating the cells.

However, additional passaging and harvesting formulations arecontemplated herein including formulations containing EDTA and EGTA,other Ca²⁺ chelators besides sodium citrate, or combinations of variousCa²⁺ chelators. Two factors are identified related to cell detachment,the Ca²⁺ chelator concentration and osmolarity. When the concentrationis too low, the bond between the cells and the matrix is not effectivelydisrupted and the formulation cannot effectively detach the cells. Whenthe concentration is too high, the bond between the cells is overlydisrupted, the size of the detached clusters decrease, and the detachedclusters can be fragile and easily disintegrated when further handled.

Not to be limited by theory, the shrinking of the cells or the coloniescaused by high osmorality can result in cell detachment, especially atthe edge of the colonies. However, the cells could also be damaged ifthe osmolarity is too high. As illustrated herein, when the osmolarityis too high, the cells are not effectively detached from the surface.EXAMPLE 4, provides studies toward further optimization. Through use ofthe experimental design disclosed herein, other cell-detaching/passagingformulations containing various Ca²⁺ chelators can be developed and areconsidered within the scope of the present invention.

EXAMPLE 4

Optimization of Sodium Citrate Formulations Towards High PlatingEfficiency After Extended Treatment

Studies were performed to determine the optimum sodium citrateformulation to be used for achieving high plating efficiency of the hESCclusters detached after extended treatment with sodium citrate solution.hESC culture was treated with solutions of varying sodium citrateconcentration (1, 5, and 10 mM) and osmolarity (400, 500, 600, 700, and800 mOsmol/L, achieved by adjusting the concentration of KCl) for 5, 10,15, and 20 minutes. At the indicated treatment time points, percentharvest, cluster size, and re-plating efficiency were compared for eachsodium citrate solution. To quantify re-plating efficiency, 2×10⁵ viablecells detached from the surface were re-plated onto one well of asix-well plate coated with Matrigel, and the attached cells wereharvested and counted the next day. Assuming that the re-attached cellsgrew to double their population within one day, the re-platingefficiency was then calculated as the number of the cells harvested thenext day divided by 4×10⁵. As shown in FIG. 28A: 1/ cluster sizedecreases with the increase of the treatment time; 2/ cluster sizedecreases with the increase of sodium citrate concentration; and 3/cluster size increases with the increase of the osmolarity. Resultsindicated that compared with all solutions tested, the low sodiumcitrate concentration solution (1 mM) with high osmolarity (600-700mOsmol/L) produced good cell detachment, large clusters and high platingefficiency even after extended treatment (FIGS. 28A-C). However, highosmolarity (>700 mOsmol/L) appeared to cause hESC cluster roll-up atdetachment, which can result in undesired morphology of the re-platedcolonies and cell death or differentiation at the centers of thosecolonies.

EXAMPLE 5

Use of Sodium Citrate in Dry Passaging

Loss of the cells can occur during treatment with cell-detachingsolutions. This is because the culture is soaked in the solution duringtreatment, and the solution is removed prior to detaching the culturewith cell culture medium. In order to eliminate this loss, studies wereperformed to find out whether the solution can be added and removedright away at the beginning of the treatment, and whether the culturecan be detached with only the residue of the solution on the culturesurface (“dry passaging”). The proposed new procedure is depicted inFIG. 22. As also indicated in FIG. 22, two treatment time points (5, 10min), two incubation conditions (Room Temperature, 37° C. incubator)were compared, and both Versene® EDTA and citrate formulation Dis2#3were tested for this dry passaging procedure. Based on the cluster sizedistributions and the images of re-plated cells, it was determined that“dry passaging” procedure works for both Versene® EDTA and citrateformulation, and the best condition based on this experiment was 5 minof incubation at room temperature. “Dry passaging” could be adopted toavoid the harvest loss in detachment solutions. Although additionaloptimization of the citrate formulation for the application in “drypassaging” to yield high detachment, desirable cluster size distributionand re-plating efficiency can be determine by one of ordinary skill inthe art, one exemplary formulation is Dis2#3.

EXAMPLE 6

Use of Sodium Citrate in Leave-In Passaging

As another alternative approach to streamline the detachment andpassaging procedure and to avoid the loss of cells at the removal of thecell-detaching solutions, “leave-in passaging” procedure was evaluated.FIG. 24 depicts the methodology used in “leave-in passaging”. Basically,after soaking the culture with the cell-detaching solution for a certaintreatment time, the solution is not removed from the culture; instead,it is left in the culture and neutralized with culture medium. The cellsare then dislodged from the surface by a swirling motion, and seededonto a fresh surface afterwards. The cell-detaching solution is left inthe final cell harvest and is later transferred into the next passage ofthe culture when the cell harvest is seeded onto fresh surface. Giventhat citrate is sometimes used as a normal component of some cellculture medium, the remaining citrate should not have deleteriouseffects on cell cultures. Such a protocol provides increased ease-of-useand scalability of the sodium citrate formulations for cell detachment.For example, the solutions are not required to be removed from theculture vessel and tapping of the vessel is not used to dislodge thecells. The protocol could be applied in small scale bench-top cultureplate culture format; it could also be applied in, for example,harvesting cells grown in 10-layer, or even 40-layer cell culturevessels that have to be handled by robots (e.g. Automatic Cell FactoryManipulator).

The study described in FIG. 24 was conducted on hESC culture grown inStemPro® medium. The culture was treated with two sodium citrateformulations, solution #3 and #13 described in EXAMPLE 4, at the volumeof 0.5 mL and 0.75 mL. FIG. 25 shows the post-detachment viable cellnumber and percent viability achieved using leave-in passaging with thetwo sodium citrate formulations. FIG. 26 shows the post-detachmentcluster size distribution and plating efficiency achieved using leave-inpassaging with the two sodium citrate formulations. The data indicatethat both sodium citrate formulations worked well and providedcomparable results; complete detachment occurred within 7-10 minutes,cell viability post-detachment was >95%, clusters were primarily large(20-75% post-detachment cells were in the format of clusters of >200 μmequidiameter; <8% were <40 μm), and the post-detachment platingefficiency was as high as around 80%. In addition, the data alsoindicate that 0.5 mL of cell-detachment citrate solution was sufficientto detach the cells, and the size of post-detachment hESC clusters wererelatively smaller when 0.75 mL solution was used.

EXAMPLE 7

Passaging of Mesenchymal Stem Cells

Studies were performed to assess the effect of sodium citrateformulations, which were optimized for hESC detachment, on detaching andpassaging MSCs. Four formulations were used and compared: Versene® EDTA,citrate Dis2#3, citrate #3 and citrate #13 (indicated as citrate 2-3, 3and 13 in FIG. 27). As shown in FIG. 27, sodium citrate solution #3provided optimum results. Treatment of MSCs with citrate #13 for 10minutes followed by tapping of the vessel was sufficient to detach 80%of the cells, and resulted in 90% post-detachment viability and the bestpost-detachment replating, compared with the other three formulations.

EXAMPLE 8

Evaluation of Sodium Citrate Formulations as Cell Detachment andPassaging Solutions in Long-Term Cultivation of hESCs

The citrate formulations described above were initially identified andoptimized for applications in harvesting and passaging hESCs grown inlarge-scale in closed culture vessels. To dislodge the cells after thetreatment with the cell-detaching solutions, culture vessels wereagitated by tapping, swirling or shaking in the experiments to simulatethe cell detachment process in closed culture vessels. In small scalebench-top cultivation of hPSCs, open culture platforms, e.g. six-wellplates or T-flasks, are often used, and cells are accessible to bedislodged by hosing with a stream of culture medium, as described inTable 1 under “EDTA method”. To evaluate the use of sodium citratesolutions in the small-scale bench-top applications, a preliminary studywas conducted to test citrate solution #3 and #13 on hESC culture insix-well plate, and compared their performance with Versene® EDTA. Theculture was grown in mTeSR1® medium and was treated for 5-8 minutes withthe formulations followed by hosing (note that for large scaleapplication, longer treatment, around 10 min, may needed). Treatment ofhESC cultures with either solution for 5 minutes followed by hosing witha pipette works well and achieves 95% harvesting and approximately70-85% replating efficiency. The replating efficiency of citratesolution #13 was about 15% higher than that of Versene® EDTA.

Long term hESCs cultures in StemPro® and mTeSR1® were initiated andmaintained for over 50 passages (about 8 months) by continuouslypassaging with citrate formulations. Sodium citrate solutions Dis2#3, #3and #13 were evaluated and Versene® EDTA was included in this study as acomparison to the citrate formulations. As shown in FIG. 29, hESCspassaged with citrate solution #13 for 31 passages in StemPro® medium,and 34 passages in mTeSR1®, respectively, stained positive for hESCmarkers, including OCT4, Sox2, Nanog, SSEA4, TRA-1-60 and TRA-1-81). Themarker expressions were further quantified with flow cytometry. 80-100%of the hESCs passaged with the citrate solution for 36 passages in^(StemPro)® , and 30 passages in mTeSR1®, respectively, stained positivefor OCT4, SSEA4, TRA-1-60 and TRA-1-81, and the percentages of positivecells were comparable to those of cells directly thawed out from thehESC bank (the starting cells for the long term culture). Thecharacterization results from both immunocytochemistry and flowcytometry shows that long-term passaging with sodium citrate formulationsupports sustainable culture, or self-renewal of hESCs.

Pluripotency of these hESCs was further evaluated for differentiationcapability. As shown in FIG. 30, hESCs passaged with citrate solutionfor 40 passages in StemPro® and 35 passages in mTeSR1®, respectively,were capable of generating embryoid bodies (EBs) and differentiatinginto all three germ layers, endoderm, mesoderm and ectoderm. Thedifferentiation capability of the long-term hESC culture in StemPro® wasfurther confirmed by the generation of teratomas of these cells inimmunodeficient SCID mice (FIG. 31). The results from EB differentiationand teratoma generation proved that hESCs passaged with citrate solutionfor numerous passages and prolonged time are still pluripotent andretain the capability of differentiating into all kinds of somaticcells.

G-banding karyotype of the long term hESC culture passaged with citratesolution was examined for abnormality. Three samples of hESCs culturedin StemPro® medium were karyotyped normal after passaged for 10 and 25passages with the citrate solution #13; in contrast, whereas threesamples of hESCs are normal after 10 passages with Versene® EDTA, oneout of three samples are abnormal and one sample had emergingabnormality after 25 passages with Versene® EDTA. HESCs cultured inmTeSR1® medium were also karyotyped normal after passaged for 29 timeswith the citrate solution #13.

The split ratio used to passage the long term hESC cultures was 1:15 to1:40 (i.e. for example, cells harvested from one well could be splitinto 15 to 40 fresh wells; in the context of hPSC cultivation, 1:40 isconsidered higher split ratio than 1:15), in both mTeSR1® and StemPro®.The cultures typically reached confluence on day 4 or day 5 after split.In traditional hPSC cultivation, where enzymatic treatment and scrapingis used to passage the cells, the split ratio is typically 1:2 to 1:6,and the culture typically reaches confluence between day 5 and day 7.Compared with the traditional passaging method, the passaging methoddisclosed herein results in much faster expansion of the culture. Thisnot only benefits manufacturing cells in large scale, but also shortensthe waiting time between experiments in bench-top studies. One concernrelated to high split ratio and fast expansion is the emergence ofabnormal karyotypes. However, as described above, the cultures in bothmTeSR1® and StemPro® maintained normal karyotype after extendedcultivation using sodium citrate formulation as passaging solution.

EXAMPLE 9

Determination of Operational Window for Selected Sodium CitrateFormulation

The time course study described in EXAMPLE 3 was conducted to determinethe operational windows for citrate solution #13. Versene® EDTA was usedas a control in this study. The experiment method was slightly modifiedfrom the study in EXAMPLE 3; the percent harvest was quantified based oncell counts instead of visual assessment. As compared with Versene®EDTA, sodium citrate solution #13 harvested more cells in shortertreatment time. There was bigger population of cells in the “>200 um”category when citrate solution was used, according to the “Cluster Size”plot. As a result, citrate solution #13 had a wider operational window(8-11.5 min) than Versene® EDTA. In fact, the “Operational Window” ofVersene presented no overlap in time frame to satisfy both requirements(“% harvest” >70%, and “clusters bigger than 40 um” >90%). The cellsdetached with citrate #13 had comparable plating efficiency with thosedetached with Versene® EDTA (data not shown). Overall, sodium citratesolution #13 outperforms Versene® EDTA

EXAMPLE 10

Detaching and Passaging Human Induced Pluripotent Stem Cells (iPSCs) andMultiple Lines of Human Embryonic Stem Cells with Sodium CitrateFormulations

In this EXAMPLE 10, sodium citrate formulations were tested on otherhESC lines (in addition to H9) and human iPSCs. Sodium citrate solution#13 was used to passage H17 and H14 hESCs grown in mTeSR1®. Unlike theH9 cultures used in the other studies, the starting cultures of H7 andH14 had many stroma or differentiated cells. The citrate solutionselectively detached and passaged mostly the undifferentiated hESCs, andleft the stroma and differentiated cells on the surface. Following threeconsecutive passages with the solution, the morphology of both the H7and H14 cultures was greatly improved with no obvious stroma ordifferentiated cells observed. It follows that the sodium citrateformulations can be used to detach and passage various hESC lines.

Citrate solution #13 can be used to detach and passage iPSCs. Even whenthe cells were transferred from MEF feeders onto Matrigel, theattachment was good when the detached cells were re-plated, and themajority of the MEF feeders was not detached and hence not subsequentlytransferred onto the fresh Matrigel surface. In this experiment, thehuman iPSC culture was also gradually adapted into defined medium(StemPro®) in five days at passage 1. It therefore follows that thecitrate formulations can be applied onto various human iPSC lines forpassaging and maintaining the established culture. The formulation canfurther be used to transfer the hPSC cells, including hESCs and humaniPSCs, from MEF feeders onto feeder-free surfaces, like Matrigel, withreduced time to clear up the remaining MEFs (compared with mechanicalpassaging methods involving scraping). Application of citrateformulations in the initial phase of human iPSC generation can helpspeed up the process of establishing the culture in feeder-free and/ordefined conditions.

EXAMPLE 11

Large-Scale Culture and Downstream Processing of hESCs Using SodiumCitrate Solutions

The study described in EXAMPLE 1 where hESCs were harvested fromtwo-layer cell culture vessels (e.g. Corning Cell Stacks) using a sodiumcitrate formulation was conducted on cells cultured in MEF-CM, and theformulation was composed of 15 mM sodium citrate, 135 mM KCl in DPBS.The current example describes the study in which citrate solution #13was used to harvest hESCs grown in mTeSR1®. Prior to scaling up theculture from six-well plate into multi-layer cell factories, citratesolutions #3 and #13 were assessed and compared with Versene® EDTA indetaching hESC from larger culture vessels, i.e. T225 flasks instead of6-well plates. Two hESC lines, H9 and H14 were used. The cultures weretreated with citrate solutions for 10 minutes and then incubated inculture medium for 2 minutes before the flasks were tapped to dislodgethe cells. The results indicated that both solutions are good forlarge-scale harvesting and provide greater harvesting than Versene® EDTA(for example, 30% higher than Versene® EDTA for H9 cells and 15-20%higher than Versene® EDTA for H14 cells) with similar cluster sizedistributions (primarily 40-200 μm) among both solutions and Versene®EDTA for both cell lines (data not shown). In detaching H14 cells,solution #13 seemed to slightly outperform solution #3 (5% more harvest,and bigger clusters with #13). Therefore, citrate solution #13 was usedin the study of scaling up hESC cultures into multi-layer cell culturevessels.

Regardless of the method used to obtain hESCs, compatible downstreamprocessing technologies are also needed to concentrate and purify thecells in the final harvest. As mentioned previously, current therapeuticcell volume reduction and purification technique using opencentrifugation technology is time consuming, introduces high processrisk, and is cost prohibitive for high volume batches. After processingwith KSep, the size distribution of the hESC clusters harvested bysodium citrate solution #13 did not change significantly, indicatingthat KSep processing was not significantly breaking up the clusters. Theviability of the cells after KSep was as high as over 80%, and theplating efficiency was over 60%, only 13% lower than the platingefficiency before KSep. The images of the Day 1 re-plated cultures alsoindicated that the hESC clusters harvested with the citrate solutionwere not fully disintegrated into single cells or smaller clusters byKSep processing. Overall, sodium citrate solution #13 is capable ofgenerating robust clusters that survive kSep®, automated vialing andcryopreservation with Controlled-Rate Freezer, retain high viability andplating efficiency, and present normal karyotype post-scaling up.

Having now described a few embodiments of the invention, it should beapparent to those skilled in the art that the foregoing is merelyillustrative and not limiting, having been presented by way of exampleonly. Numerous modifications and other embodiments are within the scopeof one of ordinary skill in the art and are contemplated as fallingwithin the scope of the invention and any equivalent thereto. It can beappreciated that variations to the present invention would be readilyapparent to those skilled in the art, and the present invention isintended to include those alternatives. Further, since numerousmodifications will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationillustrated and described, and accordingly, all suitable modificationsand equivalents may be resorted to, falling within the scope of theinvention.

What is claimed is:
 1. A formulation for harvesting and subsequentpassaging of human pluripotent stem cells or human mesenchymal stemcells comprising: sodium citrate, wherein said sodium citrate is at aconcentration of 1-15 mMol/Liter; a salt, said salt comprising KCL at aconcentration of 203-316 mMol/Liter and a phosphate-buffered salinesolution, wherein the phosphate-buffered saline solution isCa2+/Mg2+-free Dulbecco's phosphate buffered saline (DPBS); wherein saidformulation has an osmolarity of 418-570 mOsmol/Liter; and wherein saidformulation is a non-enzymatic cell detachment solution.
 2. Theformulation of claim 1, wherein the human pluripotent stem cells areselected from the group consisting of embryonic stem cells and inducedpluripotent stem cells.
 3. The formulation of claim 1, wherein the saltfurther comprises a salt selected from the group consisting of NaCl,Na₂HPO₃, NaH₂PO₃, K₂HPO₃, KH₂PO₃, and NaHCO₃.
 4. The formulation ofclaim 1, wherein the formulation is pH buffered with bicarbonate,phosphate, ethanolamine, triethanolamine, or trometamol.
 5. Theformulation of claim 1, wherein the formulation has a pH between7.2-7.8.
 6. A method for harvesting and subsequent passaging of humanpluripotent stem cells (hPSCs)cultured in vitro comprising: incubatingthe hPSCs in a formulation of claim 1 in cell culture plates or vesselsfor about 2-20 minutes, wherein said hPSCs detach from the cell cultureplates or vessels in clusters having cell viability between about85-100%, and wherein the average cluster size is about 10-1000 μm. 7.The method of claim 6, wherein the cell culture plates or vessels areselected from the group consisting of petri dishes, multi-well cellculture plates, stacked cell culture apparatus, and cell culturefactories.
 8. The method of claim 6, wherein the average cluster size isabout 40-500 μm.
 9. The method of claim 6, further comprising:downstream processing of clusters, wherein downstream processing isselected from the group consisting of continuous counter-flowcentrifugation technology, formulation, automated vialing andcryopreservation.
 10. A method for harvesting and subsequent passagingof human pluripotent stem cells (hPSCs) comprising: plating the hPSCs inmedium, aspirating spent medium, washing with DPBS, adding theformulation of claim 1, removing the formulation of claim 1, incubating,adding culture media, and collecting detached clusters.
 11. The methodof claim 6, wherein the formulation of claim 1 is not removed.